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WO2025033530A1 - Marqueur de diagnostic pour le cancer du côlon - Google Patents

Marqueur de diagnostic pour le cancer du côlon Download PDF

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Publication number
WO2025033530A1
WO2025033530A1 PCT/JP2024/028621 JP2024028621W WO2025033530A1 WO 2025033530 A1 WO2025033530 A1 WO 2025033530A1 JP 2024028621 W JP2024028621 W JP 2024028621W WO 2025033530 A1 WO2025033530 A1 WO 2025033530A1
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gene
linc01869
seq
nucleotide residues
nucleotide
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Japanese (ja)
Inventor
光昭 西岡
勇希 國宗
雅季 児玉
隆弘 山▲崎▼
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Yamaguchi University NUC
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Yamaguchi University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention relates to a method for determining whether or not a subject is suffering from colorectal cancer by using as an indicator the methylation level of CpG sites in the promoter region of the LINC01869 gene and/or the expression level of a transcription product of the LINC01869 gene in a biological sample collected from the subject, and a kit for use in said method.
  • fecal occult blood tests are the main screening test for colorectal cancer.
  • the effectiveness of fecal occult blood tests for colorectal cancer screening has been proven in randomized controlled trials, the attendance rate is problematic, being less than half. Possible reasons for the low attendance rate include low public awareness, as well as the hassle of collecting stool samples multiple times. For this reason, there is a need for a simpler screening test for colorectal cancer to replace the fecal occult blood test, but at present none is in practical use.
  • Patent Document 1 As a diagnostic marker for colon cancer, for example, lipocalin-type prostaglandin D2 synthase has been reported (Patent Document 1). It has also been reported that the onset of colon cancer can be predicted by detecting the cancer-related gene mina53 (Patent Document 2). It has also been reported that Lynch syndrome colon cancer can be predicted by detecting mutations in the ITGB3BP gene (Patent Document 3). However, the relationship between the methylation level of CpG sites in the promoter region of the LINC01869 gene and the expression level of the transcription product of the gene and colon cancer has not been known until now.
  • the objective of the present invention is to provide a relatively simple method for early and accurate diagnosis of colorectal cancer, as well as a kit for use in said diagnosis.
  • lncRNA long noncoding RNA
  • LINC01869 gene long noncoding ribonucleic acid
  • a method for determining colon cancer comprising the following steps (a) and (b): (a) measuring the methylation level of one or more CpG sites in the promoter region of the LINC01869 gene or the expression level of a transcription product of the LINC01869 gene in a biological sample collected from a subject; (b) determining whether or not the subject is affected with colorectal cancer using the methylation level of the CpG site or the expression level of the transcript measured in step (a) as an index; [2] A polynucleotide comprising a promoter region of the LINC01869 gene, the promoter region comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:2 and a complementary sequence thereof; the one or more CpG sites are one or more nucleotide residues selected from the group consisting of nucleotide residues 942 to 943, nucleot
  • step (a) The method of determining the expression level of the transcription product of the LINC01869 gene according to [1] above, wherein in step (a), the expression level of a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO:1 and its complementary sequence is measured.
  • the kit comprising a primer set consisting of a forward primer and a reverse primer, or a probe, for detecting methylation of one or more CpG sites in the promoter region of the LINC01869 gene, or a primer set consisting of a forward primer and a reverse primer, or a probe, for detecting expression of a transcription product of the LINC01869 gene.
  • the one or more CpG sites are one or more nucleotide residues selected from the group consisting of nucleotide residues 942 to 943, nucleotide residues 968 to 969, nucleotide residues 1003 to 1004, nucleotide residues 1017 to 1018, nucleotide residues 1044 to 1045, nucleotide residues 1072 to 1073, nucleotide residues 1132 to 1133, and nucleotide residues 1146 to 1147 in the nucleotide sequence of SEQ ID NO:2.
  • the promoter region of the LINC01869 gene after bisulfite conversion is a polynucleotide comprising a nucleotide sequence having 90% or
  • one or more CpG sites are nucleotide residues 968 to 969 and nucleotide residues 1003 to 1004 of the nucleotide sequence of SEQ ID NO: 2; a forward primer and a reverse primer for detecting methylation of one or more CpG sites in the promoter region of the LINC01869 gene anneal upstream of nucleotide residues 968 to 969 and downstream of nucleotide residues 1003 to 1004 in the promoter region of the LINC01869 gene after treatment with AciI,
  • the forward primer is a polynucleotide having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO:9
  • the reverse primer is a polynucleotide having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 10; or one or more CpG sites are at nucleotide residues 1146-1147 of the nucleotide sequence of
  • a forward primer and a reverse primer for detecting the expression of a transcription product of the LINC01869 gene anneal to the upstream and downstream sides of the transcription product of the LINC01869 gene, respectively;
  • a probe for detecting the expression of a transcription product of the LINC01869 gene hybridizes to the transcription product of the LINC01869 gene;
  • the transcription product of the LINC01869 gene is a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:1 and its complementary sequence.
  • a method for preparing data for diagnosing colorectal cancer which comprises the above-mentioned step (a) and a step (B) for preparing data for diagnosing whether or not the subject is affected with colorectal cancer, using the methylation level of the CpG site or the expression level of the transcript measured in the step (a) as an index; a method for diagnosing colorectal cancer, which comprises the above-mentioned steps (a) and (b); and a method for treating colorectal cancer or a method for preventing the worsening of colorectal cancer, which comprises the above-mentioned steps (a) and (b) and also comprises a step of administering a treatment for treating colorectal cancer or a treatment for preventing the worsening of colorectal cancer to a subject determined in step (b) to be highly likely to be affected with colorectal cancer (e.g., pharmacotherapy using an anticancer agent or the like, radiation therapy, surgical treatment,
  • pharmacotherapy using an
  • the present invention can accurately determine not only advanced colon cancer but also early stage colon cancer (stage I colon cancer), which will contribute to the treatment of colon cancer and the prevention of the progression of the disease by providing appropriate treatment to more colon cancer patients or by providing appropriate measures to prevent the worsening of colon cancer.
  • stage I colon cancer early stage colon cancer
  • Figure 1A shows the results of analyzing the expression levels of lncRNA of the LINC01869 gene in colon tumor tissues ("T” in the figure) and non-tumor colon tissues ("N” in the figure). "***” in the figure indicates that there is a statistically significant difference (p ⁇ 0.0001) based on the Mann-Whitney U test.
  • Figure 1B shows the ROC (Receiver Operating Characteristic) curve created based on the results of Figure 1A. This figure shows the results of analyzing the expression levels of lncRNA of the LINC01869 gene in colon tumor tissues and non-tumor colon tissues ("N” in the figure) at four stages (stage I, stage II, stage III, and stage IV).
  • FIG. 4B shows the results of analyzing the methylation levels of six CpG sites (No. 1 to No.
  • FIG. 1 shows the results of analyzing the methylation levels (average values) of eight CpG sites (No. 1 to No. 8 in the figure) in the promoter region of the LINC01869 gene for colon tumor tissues ("T" in the figure) and non-tumor colon tissues ("N” in the figure) derived from colon cancer patients 1 to 4.
  • Figure 6A shows the results of amplifying a region containing CpG site No.
  • FIG. 7B shows an ROC curve created based on the results of FIG. 7A.
  • "*" indicates that there is a statistically significant difference (p ⁇ 0.05) by two-sample t-test.
  • FIG. 8B shows an ROC curve created based on the results of FIG. 8A.
  • the determination method of the present invention includes the following steps: (a) measuring 1) the methylation level of one or more CpG sites in the promoter region of the LINC01869 gene, and/or 2) the expression level of a transcription product of the LINC01869 gene in a biological sample collected from a subject; and step (b) of determining whether or not the subject is suffering from colorectal cancer using the methylation level of the CpG site or the expression level of the transcript measured in step (a) as an index.
  • the method for diagnosing colorectal cancer (hereinafter, sometimes referred to as "the present determination method") is not particularly limited as long as it sequentially comprises the steps of: determining whether or not the subject is suffering from colorectal cancer using the methylation level of the CpG site or the expression level of the transcript measured in step (a) as an index; and the present determination method is a method for assisting a doctor in diagnosing the presence or absence of colorectal cancer, and does not include the act of diagnosis by a doctor.
  • the kit of the present invention includes: The purpose specified is "to be used in the present judgment method” 1) a primer set consisting of a forward primer and a reverse primer for detecting methylation of one or more CpG sites in the promoter region of the LINC01869 gene (hereinafter sometimes referred to as the "present primer set for methylation detection"), or a probe for detecting methylation of one or more CpG sites in the promoter region of the LINC01869 gene (hereinafter sometimes referred to as the "present probe for methylation detection”); and/or 2) a primer set consisting of a forward primer and a reverse primer for detecting expression of a transcription product of the LINC01869 gene (hereinafter sometimes referred to as the "present primer set for transcription product detection"), or a probe for detecting expression of a transcription product of the LINC01869 gene (hereinafter sometimes referred to as the "present probe for transcription product detection”);
  • the present kit is not particularly limited as long as it is
  • the present kit is a use invention relating to a kit for diagnosing colorectal cancer or a kit for assisting in the diagnosis of colorectal cancer, and these kits usually contain components generally used in this type of kit, such as a carrier, a pH buffer, and a stabilizer, as well as an instruction manual, a manual for diagnosing colorectal cancer, and other attached documents.
  • colon cancer refers to any cancer that occurs in the colon and/or rectum, and examples include stage I colon cancer (i.e., cancer is confined to the muscularis basement [muscle layer] of the colon), stage II colon cancer (i.e., cancer has spread beyond the muscularis basement of the colon), stage III colon cancer (i.e., cancer has metastasized to lymph nodes regardless of the depth of invasion), and stage IV colon cancer (i.e., cancer has metastasized to organs distant from the colon, such as the liver, lungs, and peritoneum).
  • stage I colon cancer i.e., cancer is confined to the muscularis basement [muscle layer] of the colon
  • stage II colon cancer i.e., cancer has spread beyond the muscularis laminate of the colon
  • stage III colon cancer i.e., cancer has metastasized to lymph nodes regardless of the depth of invasion
  • stage IV colon cancer i.e., cancer has metastasized to organs distant from the colon, such as the liver, lungs, and peritoneum
  • a “biological sample” may be any sample derived from a subject (living body), and examples include blood samples (e.g., plasma samples, serum samples), colon tissue samples, urine samples, etc.
  • the promoter region of the LINC01869 gene specifically includes a polynucleotide (i.e., a double-stranded polynucleotide) that includes a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:2 and its complementary sequence.
  • methylation of a CpG site means the addition of a methyl group to the 5th carbon of a cytosine (C) in a dinucleotide (CpG) site represented by 5'-CG-3' in a nucleotide sequence in DNA (deoxyribonucleic acid).
  • the "one or more CpG sites” specifically refer to one or more CpG sites selected from the group consisting of nucleotide residues 58 to 59, nucleotide residues 203 to 204, nucleotide residues 225 to 226, nucleotide residues 253 to 254, nucleotide residues 338 to 339, nucleotide residues 352 to 353, nucleotide residues 370 to 371, nucleotide residues 425 to 426, nucleotide residues 942 to 943, nucleotide residues 968 to 969, nucleotide residues 1003 to 1004, nucleotide residues 1017 to 1018, nucleotide residues 1044 to 1045, nucleotide residues 1072 to 1073, nucleotide residues 1132 to 1133, and nucleotide residues 1146 to 1147 in the nucleotide sequence of SEQ ID NO: 2.
  • nucleotide residues can be 2 or more nucleotide residues, and the effect of which is demonstrated in the present Example described later, so that in the nucleotide sequence of SEQ ID NO: 2, one or more nucleotide residues selected from the group consisting of nucleotide residues 942-943 (CpG site No. 1 in Table 3), nucleotide residues 968-969 (CpG site No. 2 in Table 3), nucleotide residues 1003-1004 (CpG site No. 3 in Table 3), nucleotide residues 1017-1018 (CpG site No. 4 in Table 3), nucleotide residues 1044-1045 (CpG site No.
  • nucleotide residues 1072-1073 (CpG site No. 7 in Table 3), nucleotide residues 1132-1133 (CpG site No. 6 in Table 3), and nucleotide residues 1146-1147 (CpG site No. 8 in Table 3) can be preferably exemplified.
  • transcription product of the LINC01869 gene refers to an lncRNA of 200 nucleotides or more (e.g., 300-1000 nucleotides, 600-1000 nucleotides, 900-1000 nucleotides) transcribed from the LINC01869 gene, and the transcription product of the LINC01869 gene is different from mRNA that codes for a protein and non-coding RNA other than lcRNA (e.g., transfer RNA, ribosomal RNA, siRNA, small non-coding RNA such as microRNA of about 20 nucleotides, etc.).
  • transfer RNA e.g., transfer RNA, ribosomal RNA, siRNA, small non-coding RNA such as microRNA of about 20 nucleotides, etc.
  • RNA usually a single-stranded polynucleotide
  • SEQ ID NO: 1 the nucleotide sequence of SEQ ID NO: 1
  • U uracil residue
  • the method for measuring the methylation level of the CpG site in the promoter region of the LINC01869 gene may be any method capable of specifically detecting the methylation of the CpG site in the promoter region of the LINC01869 gene in a biological sample, and may be, for example, a method in which DNA is isolated and purified from a biological sample or a processed product thereof (for example, in the case of a blood sample, a serum sample or a plasma sample; in the case of a urine sample, urine from which impurities have been removed by centrifugation) according to a standard method, the genomic DNA is treated with sodium bisulfite using a commercially available kit or the like, and methylation at the CpG site is detected by a methylation specific PCR (MSP) method, a bisulfite sequencing method, or the like, after bisulfite conversion.
  • MSP methylation specific PCR
  • Methylation at the CpG site can also be measured by a DNA methylation measurement method using an ICON probe (the present methylation detection probe), which is a novel method that does not require bisulfite conversion.
  • Another example is a method of detecting methylation at a CpG site by combining methylation-sensitive restriction enzyme treatment with PCR.
  • isolated and purified DNA is treated with a methylation-sensitive restriction enzyme (e.g., AciI, AvaI, BceAI, FauI, EciI, etc.) that uses a region containing a CpG site as a restriction enzyme site (also called a "recognition sequence"), and PCR is performed using forward and reverse primers that can be amplified by PCR from the upstream and downstream sides of the CpG site, and the amount of the PCR amplified product is used as an indicator to measure methylation at the CpG site.
  • a methylation-sensitive restriction enzyme e.g., AciI, AvaI, BceAI, FauI, EciI, etc.
  • the above-mentioned MSP method is a method for analyzing the presence or absence of methylation at CpG sites by performing PCR using the promoter region of the LINC01869 gene after bisulfite conversion as a template and a primer set for detecting methylation, which is a primer set that amplifies when the CpG site to be measured is methylated (methylation-specific primer set) or a primer set that amplifies when the CpG site to be measured is not methylated (non-methylation-specific primer set).
  • cytosine (C) in the promoter region (DNA) of the LINC01869 gene to uracil (U), but this conversion reaction does not occur in methylated cytosine.
  • the promoter region of the LINC01869 gene is a polynucleotide having the nucleotide sequence of SEQ ID NO: 2 (see Table 3)
  • sodium bisulfite treatment does not convert the cytosines in the 16 CpG sites to uracil, but converts the other cytosines to uracil, resulting in a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 after bisulfite conversion (see Table 4), and when all 16 CpG sites in SEQ ID NO: 2 are not methylated, sodium bisulfite treatment converts all cytosines, including those in the 16 CpG sites, to uracil,
  • Methylation-specific primer sets can be prepared by designing the CpG sites to be measured assuming that they are in a bisulfite-unconverted state (i.e., CG), and non-methylation-specific primer sets can be prepared by designing the CpG sites to be measured assuming that they are in a bisulfite-converted state (i.e., UG). Note that in PCR, U is replaced with T, so when designing primers, T is used instead of U.
  • the CpG site being measured can be determined to be methylated, and if a PCR amplification product using the methylation-specific primers is not detected and/or a PCR amplification product using the non-methylation-specific primer set is detected, the CpG site being measured can be determined to be unmethylated.
  • the above-mentioned MSP method can be carried out, for example, using a PCR device such as a droplet digital PCR [ddPCR] device, according to the method described in the literature "J.G. Herman et al., Proc. Natl. Acad. Sci. USA 1996, 93:9821-9826", the method described in the literature “C.A. Eads et al., Nucleic Acids Res. 2000, 28:E32”, the method described in the literature “K. Rand et al., Methods 2002, 27:114-120”, the method described in the literature “D.T. Akey et al., Genomics 2002, 80:376-384", etc.
  • a PCR device such as a droplet digital PCR [dPCR] device
  • the level of methylation at CpG sites can be measured, for example, by pyrosequencing (S. Colella et al., Biotechniques 2003, 35:146-150; J. Tost et al., Biotechniques 2003, 35:152-156).
  • the above-mentioned bisulfite sequencing method involves using the promoter region of the LINC01869 gene after bisulfite conversion as a template, performing PCR using the present methylation detection primer set designed to amplify a region containing CpG sites, and subjecting the resulting PCR product to sequence analysis using a sequencer such as a next-generation sequencer to analyze the presence or absence of methylation at the CpG sites.
  • the forward primer in the present primer set for detecting methylation anneals to a complementary sequence of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, which is located upstream of a CpG site to be measured in the promoter region of the LINC01869 gene after bisulfite conversion specifically, a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and its complementary sequence
  • the reverse primer in the present primer set for detecting methylation anneals to a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, which is located downstream of a CpG site to be measured in the promoter region of the LINC01869 gene after bisulfite conversion specifically, a polynucleotide containing
  • the forward primer and the reverse primer are arranged so as to sandwich the CpG site to be measured, thereby amplifying a region containing the CpG site.
  • multiple primer sets for detecting methylation of the present invention may be used, each set sandwiching each CpG site.
  • using a primer set for detecting methylation of the present invention that sandwiches the most upstream CpG site and the most downstream CpG site is more efficient and preferable, since only one primer set is required and only one PCR is required.
  • upstream side means the side of the promoter region of the LINC01869 gene from the perspective of the LINC01869 gene
  • downstream side means the side of the LINC01869 gene from the perspective of the promoter region of the LINC01869 gene.
  • the PCR amplification products that sandwich the CpG site to be measured are sequenced, and if the target CpG site is read as "CG,” it can be determined that the target CpG site is methylated, and if the target CpG site is read as "TG,” it can be determined that the target CpG site is not methylated.
  • the method for measuring the expression level of the transcript of the LINC01869 gene may be a method of directly detecting the transcript of the LINC01869 gene itself (RNA) in the biological sample and measuring the expression level of the transcript, or a method of detecting cDNA (complementary DNA) (hereinafter sometimes referred to as "cDNA of the LINC01869 gene") synthesized by PCR using the transcript of the LINC01869 gene in the biological sample as a template and reverse transcriptase and measuring the expression level of the cDNA of the LINC01869 gene, but since the effectiveness of this method has been demonstrated in the present embodiment described below, a suitable example is a method for measuring the expression level of the transcript of the LINC01869 gene by measuring the expression level of the cDNA of the LINC01869 gene (more specifically, a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID
  • Methods for directly detecting the transcription product itself (i.e., RNA) of the LINC01869 gene in a biological sample include, for example, Northern blotting using the present transcription product detection probe, microarray, and ISH (in situ hybridization).
  • Methods for detecting the cDNA (usually a double-stranded polynucleotide) of the LINC01869 gene include, for example, PCR (e.g., real-time PCR, ddPCR) using the present transcription product detection primer set; Southern hybridization using the present transcription product detection probe, and microarray.
  • a method for determining whether or not a subject is affected with colorectal cancer can include, for example, a method for determining whether or not a subject is affected with colorectal cancer based on the criterion that the value is higher than a certain threshold value (cut-off value).
  • cut-off values include the average value of the methylation level of CpG sites in the promoter region of the LINC01869 gene or the expression level of the transcript of the LINC01869 gene in biological samples from control subjects not affected with colorectal cancer; the average value + standard deviation (SD); the average value + 2SD; the average value + 3SD; the median of the methylation level or the expression level of the transcript; the third quartile of the methylation level or the expression level of the transcript; the maximum value of the methylation level or the expression level of the transcript; and the like.
  • SD standard deviation
  • 2SD the average value + 2SD
  • + 3SD the median of the methylation level or the expression level of the transcript
  • the third quartile of the methylation level or the expression level of the transcript the maximum value of the methylation level or the expression level of the transcript; and the like.
  • the cutoff value can be calculated using an ROC curve with statistical analysis software based on data on the methylation levels of CpG sites or the expression levels of transcripts in colon cancer patients and data on the methylation levels of CpG sites or the expression levels of transcripts in control subjects who do not have colon cancer, so as to increase sensitivity (the proportion of people with colon cancer who can be correctly judged as positive) and specificity (the proportion of people without colon cancer who can be correctly judged as negative).
  • the method for determining whether or not a subject is affected by colorectal cancer specifically includes the following steps: comparing the methylation level of the CpG site in the promoter region of the LINC01869 gene in the biological sample collected from the subject with the methylation level of the CpG site in the promoter region of the LINC01869 gene in the biological sample collected from a control individual (e.g., a healthy individual) not suffering from colorectal cancer, and determining that the subject is highly likely to be suffering from colorectal cancer if the methylation level of the CpG site in the promoter region of the LINC01869 gene in the biological sample collected from the subject is higher than the methylation level of the CpG site in the promoter region of the LINC01869 gene in the biological sample collected from the control individual not suffering from colorectal cancer;
  • An example of a method involves comparing the expression level of the LINC01869 gene transcript in a biological sample collected from a subject with
  • the methylation detection primer set in the kit is more preferably one that detects one or more CpG sites selected from the group consisting of the 942-943rd CpG site, the 968-969th CpG site, the 1003-1004th CpG site, the 1017-1018th CpG site, the 1044-1045th CpG site, the 1072-1073rd CpG site, the 1132-1133rd CpG site, and the 1146-1147th CpG site in the nucleotide sequence of SEQ ID NO:2, as its effectiveness has been demonstrated in the present Example described below.
  • the primer set for detecting methylation in this case may be, for example, a primer set used in the MSP method described above (i.e., a methylation-specific primer set and/or a non-methylation-specific primer set), a primer set used in the bisulfite sequencing method described above (i.e., a primer set consisting of a forward primer and a reverse primer that anneal to the upstream and downstream sides, respectively, of the CpG site in the promoter region of the LINC01869 gene after bisulfite conversion), a primer set used in the combination of the methylation-sensitive restriction enzyme treatment and PCR described above (i.e., a primer set consisting of a forward primer and a reverse primer that anneal to the upstream and downstream sides, respectively, of the CpG site after treatment with a methylation-sensitive restriction enzyme that is not cleaved when the CpG site is methylated), etc.
  • a primer set used in the MSP method described above i.e.
  • primers examples include a primer set consisting of a forward primer (e.g., a polynucleotide having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:5) and a reverse primer (e.g., a polynucleotide having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:6) that anneal to the upstream and downstream sides, respectively, of a CpG site in the promoter region of the LINC01869 gene after bisulfite conversion (specifically, a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4 and its complementary sequence), and a primer set consisting of a forward primer and a reverse primer that anneal to the upstream and downstream sides, respectively, of a CpG site after treatment with a methylation-sensitive restriction enzyme that is not cleaved when the CpG site
  • the above-mentioned "primer set consisting of a forward primer and a reverse primer that anneal respectively to the upstream and downstream sides of the CpG site after treatment with a methylation-sensitive restriction enzyme that is not cleaved when the CpG site is methylated” includes, for example, a forward primer that anneals to the upstream side of the 968th to 969th nucleotide residues and the 1003rd to 1004th nucleotide residues of the nucleotide sequence of SEQ ID NO: 2 in the promoter region of the LINC01869 gene after treatment with AciI and is a polynucleotide having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 9; and a reverse primer that anneals to the downstream side of the 1003rd to 1004th nucleotide residues in the promoter region of the LINC01869 gene after treatment with AciI and is a polynucleotide having 90%
  • a primer set consisting of a forward primer that anneals upstream of nucleotide residues 1146 to 1147 in the promoter region of the LINC01869 gene after treatment with AvaI and is a polynucleotide having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:7; and a reverse primer that anneals downstream of nucleotide residues 1146 to 1147 in the promoter region of the LINC01869 gene after treatment with AvaI and is a polynucleotide having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:8 or 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:10; is preferred when the CpG site is nucleotide residues 1146 to 1147 in the nucleotide sequence of SEQ ID NO:2.
  • the methylation detection probe in the kit can be, for example, the ICON probe described above, and more specifically, a probe that hybridizes to the promoter region of the LINC01869 gene, including a CpG site (specifically, a polynucleotide that includes a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:2 and its complementary sequence).
  • the primer set for detecting the transcription product in the kit can be, for example, a primer set used in the PCR method described above, and more specifically, a primer set consisting of a forward primer and a reverse primer that anneal to the upstream and downstream sides, respectively, of the cDNA of the LINC01869 gene (more specifically, a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:1 and its complementary sequence).
  • the probe for detecting the transcription product in the kit can be, for example, a probe used in the Northern blotting method, microarray method, ISH method, Southern hybridization method, microarray method, etc., described above, and more specifically, a probe that hybridizes to a part or all of the cDNA of the LINC01869 gene (more specifically, a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:1 and its complementary sequence).
  • the primers and probes in this kit are typically single-stranded polynucleotides, and their length is, for example, 10 nucleotides or more (preferably 12, 14, or 16 nucleotides or more) and 50 nucleotides or less (preferably 40, 30, 28, or 26 nucleotides or less).
  • the primers and probes in this kit may be DNA, RNA, or DNA/RNA chimeras, but are preferably DNA.
  • some or all of the primers and probes may have nucleotides replaced with artificial nucleic acids such as PNA (polyamide nucleic acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, bridged nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), GNA (Glycerol nucleic acid), and TNA (Thresenucleic acid).
  • PNA polyamide nucleic acid, peptide nucleic acid
  • LNA registered trademark, locked nucleic acid, bridged nucleic acid
  • ENA registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids
  • GNA Glycerol nucleic acid
  • TNA Thresenucleic acid
  • the primer set and probe can be labeled in order to visualize and/or quantify the target transcription product, PCR product, region containing a CpG site, etc.
  • the labeling substances in the labeling substances of the primer set and the probe include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase, and acetylcholinesterase; the above-mentioned fluorescent substances; fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and luciferase; 3 H, 14
  • peroxidase
  • sequence identity of 90% or more means that the sequence identity is at least 90%, preferably 92% or more, more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, particularly preferably 96% or more, especially more preferably 97% or more, especially more preferably 98% or more, even more especially preferably 99% or more, and most preferably 100% sequence identity.
  • Example 1 Analysis of lncRNA expression of LINC01869 gene 1.
  • cDNA SEQ ID NO: 1 (see Table 1) derived from lncRNA, which is a transcription product of the LINC01869 gene
  • quantitative PCR was performed using the prepared cDNA as a template, a TaqMan Gene Expression Assay probe-primer set (Assay ID: Hs00382437_g1, Thermo Fisher Scientific), and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) with a real-time PCR device (QuantStudio 5 Real-time PCR System) (Thermo Fisher Scientific).
  • Thermo Fisher Scientific Thermo Fisher Scientific.
  • the PCR conditions are shown in 1) to 2) below.
  • the site amplified by the primer set is a 56-base-long region in exon 2 to exon 3 of the LINC01869 gene (underlined portion in Table 1).
  • cDNA derived from the mRNA of the ⁇ -actin gene ie, cDNA of the ⁇ -actin gene
  • was detected as an internal standard assay ID: 4326315E, Thermo Fisher Scientific).
  • the PCR cycle number (threshold cycle; Ct value) at which the PCR product became constant was measured, and the relative Ct value of the cDNA amplified product of the LINC01869 gene was calculated using the comparative Ct method ( ⁇ Ct method) relative to the Ct value of the cDNA amplified product of the ⁇ -actin gene as the standard. From this relative Ct value, the relative amount of cDNA of the LINC01869 gene, i.e., the expression level of the lncRNA of the LINC01869 gene, was calculated.
  • Statistical analysis was performed using StatFlex ver.7 (Artec) and JMP Pro 14 (SAS Institute Japan).
  • colon tumor tissues were classified into four stages (stage I, stage II, stage III, and stage IV), and the expression levels of LINC01869 gene lncRNA were compared with those of non-tumor colon tissues.
  • stage I stage II, stage III, and stage IV
  • the results showed that the expression levels of LINC01869 gene lncRNA were significantly reduced even between stage I colon tumor tissues (see Figure 2). This result indicates that the presence or absence of colorectal cancer can be accurately determined at an early stage using the expression level of lncRNA of the LINC01869 gene as an indicator.
  • Example 2 Methylation analysis of CpG sites in the LINC01869 gene promoter region 1 It is known that epigenetic transcriptional repression occurs when CpG sites in the promoter region of a gene are methylated. Therefore, we next analyzed the relationship between the decrease in the expression level of LINC01869 gene lncRNA and the methylation of CpG sites in the promoter region of the LINC01869 gene.
  • the bisulfite-converted DNA was used as a template to perform PCR using a primer set consisting of a forward primer (LINC1869met-F7) (SEQ ID NO: 5; 5'-TGATTTTGGAGGGTGATTAA-3') and a reverse primer (LINC1869met-R5) (SEQ ID NO: 6; 5'-CTAAATCAAAACTCCCCA-3') and TaKaRa EpiTaq HS (for bisulfite-treated DNA) (Takara Bio Inc.) in a PCR device (Veriti Thermal Cycler) (Thermo Fisher Scientific).
  • the PCR conditions are shown in 1) to 2) below.
  • PCR products were purified using a QIAEN Gel Extraction Kit (QIAGEN), and then TA cloning was performed using pGEM-T Easy Vector Systems (Promega). Ten clones were sequenced using a SeqStudio Flex Genetic Analyzer (Thermo Fisher Scientific) to analyze the methylation levels of six CpG sites (specifically, "CG” [CpG site No. 1-6] indicated by superscript numbers 1-6 in Table 3). Note that while unmethylated CpG sites are read as "TG” due to bisulfite conversion, methylated CpG sites are not converted to bisulfite and are read as "CG,” making it possible to detect the presence or absence of methylation at CpG sites.
  • CG SeqStudio Flex Genetic Analyzer
  • the two double underlined portions in the table indicate the sites to which the PCR amplification primer set (LINC1869met-F7 and LINC1869met-R5) designed for the bisulfite-converted DNA anneals (the same applies to Tables 4 and 5 below).
  • the underlined portions in the table indicate CpG sites (the same applies to Tables 4 and 5 below).
  • uracil (U) converted by sodium bisulfite treatment is shown as thymine (T) enclosed in a box for convenience (the same applies to Table 5 below).
  • Example 3 Methylation analysis of CpG sites in the LINC01869 gene promoter region 2
  • a methylation-sensitive enzyme was used to detect the methylation level of CpG sites.
  • Serum was used as a biological sample.
  • CpG site No. 8 in the LINC01869 gene promoter region has a restriction enzyme site for methylation-sensitive restriction enzyme (AvaI) (see Table 6), and CpG sites No. 2 and No. 3 in the LINC01869 gene promoter region have a restriction enzyme site for methylation-sensitive restriction enzyme (AciI) (see Table 7).
  • methylated human genomic DNA (EpiScope [registered trademark] Methylated HCT116 gDNA, manufactured by Takara Bio Inc.) and demethylated human genomic DNA (5% or less of all CpG sites are methylated) (EpiScope Unmethylated HCT116 DKO gDNA, manufactured by Takara Bio Inc.) were used as templates.
  • a methylation-sensitive restriction enzyme (AvaI or AciI), BstUI (a restriction enzyme that cleaves sites other than PCR amplified sites to fragment DNA), and exonuclease I (ExoI) (an enzyme for cleaving single-stranded polynucleotides) were added to a solution (10 ⁇ L) containing 5 ng of methylated human genomic DNA and demethylated human genomic DNA, and the mixture was incubated at 37° C. for 1 hour, followed by incubation at 65° C. for 20 minutes, at 60° C. for 8 hours, and at 98° C. for 15 minutes to inactivate the enzymes.
  • AvaI or AciI a restriction enzyme that cleaves sites other than PCR amplified sites to fragment DNA
  • ExoI exonuclease I
  • a portion of the mixture (5 ⁇ L) was used as a template to perform PCR using a primer set (see Table 6) consisting of a forward primer (LINC1869-CORD-F1 [SEQ ID NO: 7; 5'-GCATTGGTAGGTGTTGGCTTG-3']) and a reverse primer (LINC1869-CORD-R2 [SEQ ID NO: 8; 5'-GAGTCAGAGCTCCCCAGA-3']) for the AvaI-treated sample, and a primer set (see Table 7) consisting of a forward primer (LINC1869-CORD-F4 [SEQ ID NO: 9; 5'-CCTGTGCTGAGTCCTCCT-3']) and a reverse primer (LINC1869-CORD-R6 [SEQ ID NO: 10; 5'-GCCGAGGCAGAGGAAAC-3']) for the AciI-treated sample, and Absolute Q DNA Digital PCR Master Mix (5X) (Thermo Fisher Scientific) was used to perform PCR using a digital
  • the PCR was performed using a PCR PCR kit (manufactured by Biosciences Scientific) to measure the copy number of the PCR product containing CpG site No. 8 (FIG. 6A) and the copy number of the PCR product containing CpG sites No. 2 and No. 3 (FIG. 6B).
  • the PCR conditions are shown in 1) to 2) below.
  • PCR was performed on the samples before enzyme treatment according to the method described in the above "1. Preliminary Experiment” to measure the "copy number of the PCR product containing CpG site No. 8 before AvaI treatment” and the "copy number of the PCR product containing CpG site No. 2 and No. 3 before AciI treatment”. Then, the "copy number of the PCR product containing CpG site No. 8 after AvaI treatment (x 10)" relative to the "copy number of the PCR product containing CpG site No. 8 before AvaI treatment” was calculated (see FIG. 7A), and the "copy number of the PCR product containing CpG site No. 2 and No. 3 after AciI treatment (x 10)" relative to the "copy number of the PCR product containing CpG site No. 2 and No. 3 before AciI treatment” was calculated (see FIG. 8A).
  • the two double underlined parts in the table indicate the sites to which the primer set for PCR amplification (LINC1869-CORD-F1 and LINC1869-CORD-R2) anneals.
  • the nucleotide residues enclosed in boxes indicate the restriction enzyme sites of the methylation-sensitive restriction enzyme (AvaI).
  • AvaI methylation-sensitive restriction enzyme
  • the two double underlined parts in the table indicate the sites to which the primer set for PCR amplification (LINC1869-CORD-F4 and LINC1869-CORD-R6) anneals.
  • the nucleotide residues enclosed in boxes indicate the restriction enzyme sites of the methylation-sensitive restriction enzyme (AciI).
  • AciI methylation-sensitive restriction enzyme
  • the present invention contributes to the early detection, early treatment, and prevention of colon cancer progression.

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Abstract

La présente invention aborde le problème consistant à fournir : un procédé de détermination relativement facile et précise du cancer du côlon à un stade précoce ; et un kit ou analogue utilisé pour la détermination.　Dans la présente invention, il est déterminé si un sujet est atteint ou non d'un cancer du côlon à l'aide, en tant qu'indice, du niveau de méthylation d'un site CpG dans la région promotrice du gène LINC01869 et/ou du niveau d'expression d'un transcrit du gène LINC01869 dans un échantillon biologique collecté à partir du sujet.
PCT/JP2024/028621 2023-08-10 2024-08-09 Marqueur de diagnostic pour le cancer du côlon Pending WO2025033530A1 (fr)

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